Different involvement of type 1, 2, and 3 ryanodine receptors in memory processes

Abstract

The administration of the ryanodine receptor (RyR) agonist 4-Cmc (0.003-9 nmol per mouse intracerebroventricularly [i.c.v.]) ameliorated memory functions, whereas the RyR antagonist ryanodine (0.0001-1 nmol per mouse i.c.v.) induced amnesia in the mouse passive avoidance test. The role of the type 1, 2, and 3 RyR isoforms in memory processes was then evaluated by inhibiting the expression of the three RyR proteins in the mouse brain. A selective knockdown of the RyR isoforms was obtained by the i.c.v. administration of antisense oligonucleotides (aODNs) complementary to the sequence of RyR1, RyR2 and RyR3 proteins, as demonstrated by immunoblotting experiments. RyR1 (5-9 nmol per mouse i.c.v.) knockdown mice did not show any memory dysfunction. Conversely, RyR2 (1-7 nmol per mouse i.c.v.) and RyR3 (1-7 nmol per mouse i.c.v.) knockdown animals showed an impairment of memory processes. This detrimental effect was temporary and reversible, disappearing 7 d after the end of the aODN treatment. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous mobility and inspection activity, as revealed by the hole-board test. In conclusion, the lack of any involvement of cerebral RyR1 was demonstrated. These findings also showed the involvement of type 2 and type 3 RyR in the modulation of memory functions identifying these cerebral RyR isoforms as critical targets underlying memory processes.

Figure 1.

(A) Dose-response curve of 4-Cmc in the mouse passive avoidance test. 4-Cmc (0.003–9 nmol per mouse i.c.v.) was administered 30 min before the training session. Data are expressed as mean ± SEM; *P < 0.05 in comparison with vehicle-treated mice. (Vehicle) Ethanol 0.5%; (clon) clonidine 0.125 mg/kg i.p.; (diph) diphenhydramine 20 mg/kg i.p. (B) Time-course curve of 4-Cmc in the mouse passive avoidance test. 4-Cmc (0.03 nmol per mouse i.c.v.) was administered 15, 30, or 60 min before and immediately after the training session. Data are expressed as mean ± SEM; *P < 0.05 in comparison with vehicle-treated mice. (Vehicle) Ethanol 0.5%

Figure 2.

(A) Lack of effect of ryanodine in the mouse passive avoidance test. Ryanodine (0.01–1 nmol per mouse i.c.v.) was administered 15 min before the training session. Data are expressed as mean ± SEM; *P < 0.05 in comparison with saline-treated mice; (clon) clonidine 0.125 mg/kg i.p.; (diph) diphenhydramine 20 mg/kg i.p. (B) Dose-response curve of the amnesic effect of ryanodine in the mouse passive avoidance test. Ryanodine (0.0001–1 nmol per mouse i.c.v.) was administered immediately after the training session. Data are expressed as mean ± SEM; *P < 0.05 in comparison with vehicle-treated mice; (clon) clonidine 0.125 mg/kg i.p.; (diph) diphenhydramine 20 mg/kg i.p.

Figure 3.

Reversal by 4-Cmc of the amnesia induced by ryanodine in the mouse passive avoidance test. Doses administered (nmol per mouse i.c.v.) are reported below each column. 4-Cmc and ryanodine were injected 30 min before and immediately after the training session, respectively. Data are expressed as mean ± SEM; *P < 0.05 in comparison with saline-treated mice.

Figure 4.

(A) Lack of effect of anti-RyR1 treatment (5–9 nmol per mouse i.c.v.) in the mouse passive avoidance test. Doses of anti-RyR1 administered (nmol per mouse i.c.v.) are reported in each column. Data are expressed as mean ± SEM; *P < <0.05 in comparison with dODN-treated mice; (clon) clonidine 0.125 mg/kg i.p. (B) Dose-response curve of the amnesic effect of anti-RyR2 treatment (1–7 nmol per mouse i.c.v.) in the mouse passive avoidance test. Doses of anti-RyR2 administered (nmol per mouse i.c.v.) are reported in each column. Data are expressed as mean ± SEM; *P < 0.05 in comparison with dODN-treated mice; (clon) clonidine 0.125 mg/kg i.p. (C) Dose-response curve of the amnesic effect of anti-RyR3 treatment (1–7 nmol per mouse i.c.v.) in the mouse passive avoidance test. Doses of anti-RyR3 administered (nmol per mouse i.c.v.) are reported in each column. Data are expressed as mean ± SEM; *P < 0.05 in comparison with dODN-treated mice; (clon) clonidine 0.125 mg/kg i.p.

Figure 5.

Reduction of RyR1, RyR2, and RyR3 protein expression in mouse muscle, heart, cortex, and hippocampus by aODN treament in comparison with corresponding dODN-treated mice. (A) Samples (100 μg protein/lane) of microsomal fractions of mouse cortex and hippocampus, and samples of heart (20 μg protein/lane) and muscle (50 μg protein/lane) were resolved on 6% SDS-PAGE, transferred to nitrocellulose, and probed with RyR1, RyR2, or RyR3 antibody. (B) Densitometric quantitation of immunoreactive protein expressed relative to control. Data are expressed as mean ± SEM of band intensities from each of the three groups (n = 7 per group). *P < 0.05 vs. control.

Figure 6.

Reversibility of RyR1, RyR2, and RyR3 protein knockdown and memory impairment in aODN-treated mice in comparison with corresponding dODN-treated mice. (A) Samples (100 μg protein/lane) of microsomal fractions of mouse cortex and hippocampus were resolved on 6% SDS-PAGE, transferred to nitrocellulose, and probed with RyR1, RyR2, or RyR3 antibody. (B) Lack of effect of anti-RyR1 (9 nmol per mouse i.c.v.), anti-RyR2 (7 nmol per mouse i.c.v.), and anti-RyR3 (7 nmol per mouse i.c.v.) in the mouse passive avoidance test performed 7 d after the end of the aODN repeated treatment. Data are expressed as mean ± SEM; *P < 0.05 in comparison with the corresponding training session values (Student’s t-test).

Figure 7.

Lack of effect of anti-RyR1 (9 nmol per mouse i.c.v.), anti-RyR2 (7 nmol per mouse i.c.v.), anti-RyR3 (7 nmol per mouse i.c.v.), ryanodine (0.1 nmol per mouse i.c.v.), and 4-Cmc (9 nmol per mouse i.c.v.) on mouse spontaneous mobility (A) and exploratory activity (B) evaluated in the mouse hole board test, and on motor coordination (C) evaluated in the rota rod test. ODNs were administered once daily for 3 d. The test was performed 18 h after the last injection. Amphetamine (amph) was administered at the dose of 1 mg/kg s.c.; data are expressed as mean ± SEM; *P < 0.05 vs. control group.